This program explores innate anti-microbial defense and inflammatory mechanisms involving the generation of reactive oxygen species (ROS). Neutrophils and other circulating phagocytes generate high levels of ROS in response to infectious or inflammatory stimuli in a process known as the respiratory burst. This response is attributed to the activity of NADPH oxidase, which produces superoxide, a precursor of ROS that are important microbicidal agents and mediators of inflammation. Patients with chronic granulomatous disease (CGD) suffer from NADPH oxidase deficiencies, resulting in enhanced susceptibility to microbial infections and aberrant inflammatory responses. This project explores the cellular mechanisms regulating the respiratory burst oxidase in phagocytes (phox system) and is characterizing related oxidant-generating NADPH oxidases expressed in non-immune cells (Nox/ Duox family of NADPH oxidases). We are characterizing sources of reactive oxygen species in non-myeloid tissues, notably colon, kidney, thyroid and salivary glands, mucosal surfaces, brain, and vascular tissues. Recent evidence from this lab indicates that several of these oxidases also serve in host defense and inflammatory processes, since they are expressed predominately on apical surfaces of epithelial cells and are induced or activated by pro-inflammatory cytokines or recognition of microbial factors. The reactive oxidants produced by these enzymes also provide redox signals that can alter gene expression patterns during differentiation, cellular senescence, programmed cell death (apoptosis), oxygen sensing, or responses to growth factors, cytokines, or hormones. [unreadable] [unreadable] Nox1 and Nox3, the closest homologues of the phagocytic (Nox2-based) oxidase, were shown to function as multi-component phox-like enzymes. We systematically examined structure-function requirements for assembly and activation of these novel oxidases. Both oxidases involve p22phox as a subunit that stabilizes these oxidases and is required for their targeting to the plasma membrane. Nox1 functionally replaces gp91phox, restoring stimulus-dependent superoxide release in cells co-expressing the cytosol factors p47phox and p67phox. We recently identified unique, colon-specific homologues of these cytosolic regulators, showing that Nox1 is a regulated, phox-like complex that can act in host defense and inflammatory processes in the colon epithelium. We also examined structural requirements for the activation and sub-cellular localization of Nox1 and Nox3-supportive components. We obtained evidence indicating both Nox1 and Nox3 function as Rac1-dependent enzymes, in which Rac1 functions in a GTP-dependent manner through its binding partner, Nox activator 1 (Noxa1) or p67phox, like the phagocytic system. Membrane targeting of the Noxa1 was shown to be dependent on the Nox organizer (Noxo1), homologous to p47phox. However, Noxo1 is constitutively bound to the membrane even without cell stimulation, principally through lipid interactions with its PX domain. We compared the subcellular distribution of four alternatively-spliced isoforms of Noxo1, along with their ability to support Nox1 activity. Noxo1 alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1 alpha and delta localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Highest Nox1 activity correlates with Noxo1 beta plasma membrane binding in most transfected cell models, while Noxo1 gamma may support oxidases targeted to the nucleus. The results indicate the variant PX domains are unique determinants of Noxo1 isoform localization and oxidase function. [unreadable] [unreadable] In efforts aimed at exploring functional roles of the renal oxidase (renox or Nox4), we are characterizing four mouse strains in which the Nox4 gene is deleted. We are investigating the proposed role of Nox4 in renal oxygen sensing and erythropoiesis, since ROS are thought to provide negative feedback signals regulating renal erythropoietin synthesis. The renal oxidase is a constitutively active enzyme, consistent with its proposed role as an oxygen-sensing enzyme. Nox4 levels were shown to respond directly to hypoxia in renal cells. Furthermore, the Nox4 promoter fused to Nox4 cDNA or to other reporters demonstrated direct responsiveness to hypoxia. Surprisingly, Nox4-/- mice exhibit a normal phenotype in the unstressed state. The hematology as well as serum and urine chemistries (i.e., urine hydrogen peroxide levels) are normal in these animals. Related studies are focused on alterations in other oxidant generating or scavenging systems to explain the mechanisms maintaining normal redox homeostasis in these Nox4-deficient mice. [unreadable] [unreadable] We are exploring the functional expression of dual oxidases (Duox1 and Duox2) in epithelial cells of airways (tracheal and bronchial surfaces), salivary gland ducts, and the gastro-intestinal tract. Their expression on apical surfaces of airway epithelium cells indicates these oxidases serve as sources of hydrogen peroxide supporting the well-documented anti-microbial (bacterial, viral, and fungal) activities of lactoperoxidase on mucosal surfaces. We are examining airway epithelial cell Duox expression in response to differentiation, exposure to microbial pathogens, and pro-inflammatory cytokines. We have confirmed that airway Duox isozymes are induced by interferon-gamma, IL-4, and IL-13, suggesting roles in airway viral and microbial infection and in inflammatory disease processes (asthma). Active recombinant forms of Duox, co-expressed with their essential maturation factors, have also been produced in whole transfected cells, which secrete hydrogen peroxide into the extracellular medium in response to calcium signals. The purified recombinant Duox2 peroxidase homology domain does not contain detectable heme, rather, it appears to have a direct role in the conversion of superoxide to hydrogen peroxide within the extracellular environment. Primary cultured human airway (bronchial) and monkey salivary gland epithelial cells were shown to produce extracellular hydrogen peroxide in a Duox-dependent manner in response to physiological agonists that trigger calcium release. These primary culture systems are being developed to confirm roles of these oxidases in anti-microbial defenses and inflammatory processes on mucosal surfaces. [unreadable] [unreadable] Finally, our long-standing interests in the phagocytic (Nox2-based or phox) oxidase are currently focused on the activating roles of cytosolic regulators, including p40phox and cytosolic phospholipase A2 (cPLA2). cPLA2 is recruited to the membrane during cellular activation through direct interactions with the oxidase complex, as it appears to serve an essential role in releasing arachidonic acid within the immediate environment of the newly assembled oxidase. We are also exploring recruitment of p40phox to membranes in response to cellular activation and the role of its PX domain in membrane binding.